It is becoming increasingly clear that the tumor microenvironment plays a key role in tumor progression, pointing to a need to develop technologies to study tumor cell behavior in different microenvironments. Toward this goal, we have created Valve Enabled Cell Co-Culture (VEC3) platforms, which are a new class of microfluidic devices designed for analyzing interactions between tumor cells and others in the tumor microenvironment. The technology enables separate culture of distinct cell types and cell-cell interactions through either soluble factors or physical contacts between spatially separated cell populations while maintaining fluidic control over their individual culture environment. In addition, through selective blockage of the exchange of specific ligands between distinct cell populations, the platform can be used to identify the functions of relevant ligands of interest. Traditional cell co-culture techniques and reported microfluidic cell co-culture platforms have limitations and cannot address all important cell co-culture needs. The proposed VEC3 cell co-culture platform, through the introduction of a simple, robust, and user-friendly pneumatically or hydraulically controlled valve to reversibly separate or connect adjacent cell culture chambers, not only allows for separate culture and treatment of individual cell types, but also permits real-time, live-cell imaging of cellular interactions. To date, as a proof of principle, VEC3 has been applied to observe dynamically synapse formation between hippocampal neurons, analyze tumor-endothelial interactions in normoxic and hypoxic environments, study tumor-fibroblasts interactions in 3D matrices, and quantify tumor-endothelial cross migration mediated by various molecules. In the proposed research we will further develop VEC3 through quantitative characterizations of cellular microenvironments, including cell density and uniformity, glucose and oxygen concentration, and cell-cell interaction rates. Through improved design and performance engineering, we will develop optimized platforms and operation protocols to increase the success rate of VEC3-based assays. In addition, we will implement new functions such as controlled cell-cell interactions via blockage of the exchange of specific ligands between two cell populations. More importantly, we will apply VEC3 to study tumor- endothelial cross migration mediated by various ligands and receptors and identify the functions of specific ligands in cell-cell interactions. Therefore, successful execution of the proposed research will lead to a new class of versatile, multifunctional VEC3 microfluidic platforms that are widely applicable to cancer biology. This device will be used to elucidate the molecular mechanisms underlying tumor angiogenesis, intravasation and metastasis, which could eventually lead to better cancer treatments. The overall quantitative milestone is to achieve 95% success rate in assays using the VEC3 platforms for tumor angiogenesis, intravsation and metastasis studies with quantitative parameters of cellular communication.